ABSTRACT Von Willebrand factor (VWF) synthesized in endothelial cells and megkaryocytes is a multimeric glycoprotein that mediates initial platelet tethering to subendothelium exposed at the sites of vessel injury. Upon stimulation, endothelial cells release ultra-large (UL) forms of VWF multimers that are hyperactive and prothrombotic. Upon secretion, ULVWF is rapidly cleaved by ADAMTS-13 to smaller multimers that are hemostatically active, but no longer prothrombotic. The circulating plasma VWF (pVWF) multimers, therefore, bind platelets only after activation by high shear stress or modulators. However, whether ULVWF structurally differs from pVWF remains debatable. It is also unclear as how shear stress activates pVWF. We hypothesize that 1) ULVWF differs from pVWF in their thiol-disulfide states with regards to specific cysteine residues in the C-domain; 2) shear stress activates pVWF by inducing a thiol-disulfide exchange to facilitate covalent lateral association and to form fibrillar structures; and 3) ADAMTS-13 acts as a reductase to prevent this thiol-disulfide exchange, thus VWF activation. These hypotheses are supported by our preliminary data. We will test the hypotheses through two specific aims. Aim 1 is to define the mechanism of VWF activation by thiol-disulfide exchange by several technical means. We will identify specific cysteine residues critical for the shear-induced activation of pVWF, determine whether shear stress induces the covalent lateral association of pVWF multimers as a means of activation, and detect the unfolding of VWF domains by physical forces and strength of pVWF bonds with GP Ib before and after shear exposure. Aim 2 is to define the reductase activity of ADAMTS-13 observed in our preliminary study by studying the key aspects of this activity. We will map the region(s) and specific cysteine residues involved in this reductase activity, directly measure the reduction of (UL)VWF by ADAMTS-13 under static and flow conditions, and identify cysteine residues in the pVWF C domain that are the substrate for the reductase activity. Finally, we will investigate how this reductase activity regulates the rate of VWF domain unfolding. Information generated through these experiments is critical to dissect the mechanisms of shear-induced VWF activation and to characterize this new ADAMTS-13 activity. Knowledge gained through the studies will help us to identify new strategies to regulate VWF's roles in hemostasis and thrombosis.